Assay Device With Shared Zones

ABSTRACT

Disclosed is an assay device for determining the presence and/or extent of one or more analytes in liquid sample containing a) first and second assays each comprising a flow-path having a detection zone for immobilising a labelled binding reagent, wherein detection of a labelled binding reagent at one or both detection zones is indicative of the presence and/or extent of one or more analytes; b) a shared reference zone; c) one or more light sources to illuminate the detection zones and the reference zone; d) one or more photodetectors to detect light from the detection zones and the reference zone, which photodetector/s generate a signal, the magnitude of which signal is related to the amount of light detected; and e) signal processing means for processing signals from the photodetector/s.

RELATED APPLICATIONS

The present application claims benefit of priority to U.S. ProvisionalPatent Application No. 60/991,543, filed on Nov. 30, 2007; Great BritainApplication 0809994.7, filed May 31, 2008; and Great Britain Application0717045.9, filed Sep. 1, 2007, the contents of which are incorporated byreference herein in their entireties.

FIELD OF THE INVENTION

The present invention relates to an assay device, kit and method fordetermining the presence or extent of an analyte. In particular itrelates to the determination of an analyte over an extendedconcentration range.

BACKGROUND OF THE INVENTION

Simple lateral flow immunoassay devices have been developed andcommercialised for detection of analytes in fluid samples, see forexample EP291194. Such devices typically comprise a porous carriercomprising a dried mobilisable labelled binding reagent capable ofbinding to the analyte in question, and an immobilised binding reagentalso capable of binding to the analyte provided at a detection zonedownstream from the labelled binding reagent. Detection of theimmobilised labelled binding reagent at the detection zone provides anindication of the presence of analyte in the sample.

Alternatively, when the analyte of interest is a hapten, the immunoassaydevice may employ a competition reaction wherein a labelled analyte oranalyte analogue competes with analyte present in the sample for animmobilised binding reagent at a detection zone. Alternatively the assaydevice may employ an inhibition reaction whereby an immobilised analyteor analyte analogue is provided a detection zone, the assay devicecomprising a mobilisable labelled binding reagent for the analyte.

An assay device may determine more than one analyte. For example in thecase of assays for the determining the presence of drugs of abuse, thedevice may be capable of determining a whole panel of drugs. Suchlateral flow immunoassay devices are provided with multiple detectionzones, such zones being provided on a single or multiple lateral flowcarriers.

Determination of the result of the assay has been traditionally carriedout by eye. However such devices require the result to be interpreted bythe user which introduces an undesirable degree of subjectivity.

As such, digital devices have been developed comprising an opticaldetection means arranged to determine the result of the assay as well asa display means to display the result of the assay. Digital assayreaders for use in combination with assay test-strips for determiningthe concentration and/or amount of analyte in a fluid sample are knownas are assay devices comprising an integral digital assay reader.

Light from a light source, such as a light emitting diode (LED), isshone onto a portion of the porous carrier and either reflected ortransmitted light is detected by a photodetector. Typically, the readerwill have more than one LED to illuminate various zones of the carrier,and a corresponding photodetector is provided for each of the pluralityof LEDs. EP1484601 discloses an optical arrangement for a lateral flowtest strip digital reading device comprising a baffle arrangementallowing for the possibility of reducing the number of photodetectors inthe device.

Such devices are often designed to be single use and therefore it isdesirable to keep the costs of such devices as low as possible,especially where expensive optical and electronic components areinvolved.

SUMMARY OF THE INVENTION

It is an object according to an aspect of the invention to provide anassay device having one or more shared zones enabling a reduction in thenumber of optical components that are required for an assay devicecomprising two or more assay flow-paths.

According to a first aspect, the invention provides an assay device fordetermining the presence and/or extent of one or more analytes in aliquid sample comprising:

a) first and second assays each comprising a flow-path having adetection zone for immobilising a labelled binding reagent, whereindetection of a labelled binding reagent at one or both detection zonesis indicative of the presence and/or extent of one or more analytes;

b) a shared reference zone;

c) one or more light sources to illuminate the detection zones and thereference zone;

d) one or more photodetectors to detect light from the detection zonesand the reference zone, which photodetector/s generate a signal, themagnitude of which signal is related to the amount of light detected;and

e) signal processing means for processing signals from thephotodetector/s.

It is a further object of the invention to provide an assay reader foruse with one or more assay test-strips comprising two or more assayflow-paths, the assay reader having a reduction in the number of opticalcomponents that are typically required.

According to a second aspect, the invention provides an assay reader forreading the result of first and second assays each comprising aflow-path, each flow-path comprising a detection zone for immobilisinglabelled binding reagent, wherein detection of labelled binding reagentat one or both detection zones is indicative of the presence and/orextent of one or more analytes, and a shared reference zone; said assayreader comprising:

-   -   a) one or more light sources to illuminate the detection zones,        and the shared reference zone;    -   b) one or more photodetectors to detect light from the detection        zones and the reference zone, which photodetector/s generate a        signal, the magnitude of which signal is related to the amount        of light detected; and

signal processing means for processing signals from the photodetector/s,wherein the signal obtained from the shared reference zone is used tocompensate the values of the signals obtained from the detection zones.

The first and/or second assay may comprise a labelled binding reagentprovided in a mobilisable form upstream from the detection zone in a drystate prior to use of the device.

The shared reference zone may be comprised as part of either the firstor second assay. Alternatively the reference zone may be provided on asubsidiary flow-path to the first and second assay. The reference zonemay be chosen from a portion of the flow-path not corresponding to adetection zone, or, where a dried labelled reagent is present upstreamfrom the detection zone, a portion not corresponding to where the driedlabelled reagent is present. The reference zone may be provideddownstream or upstream from the detection zone. Measurement of thereference zone enables measurement of the background levels of reflectedor transmitted light from the flow-path. The background level may beaffected by, for example, the optical reflectance of the porous carrier,the presence of liquid sample, or of components of the assay such as alabelled binding reagent. The levels of light measured at the detectionzone may therefore be corrected with respect to the levels of backgroundlight to provide a compensated signal more accurately indicative of theamount of labelled binding reagent present at the detection zone.Measurement at the reference zone also compensates for any variationbetween fluid samples applied to assay devices, for example urinesamples may vary widely in colour. The value of the signal obtained atthe reference zone for one assay is used to compensate the value of thesignal obtained at the detection zone for the other assay. As such thereference zone is “shared” between both assays. The provision of ashared reference zone can reduce the number of components required forthe assay device, since each reference zone would typically require alight source.

The concept of a shared reference zone is rather counter-intuitive. Thepurpose of a reference measurement is to allow for variations in thebackground readings of signals which can arise, inter alia, as a resultof variations in reagent or assay strip composition. Accordingly, thenormal practice is to use a separate reference zone on each assay, sothat a “dedicated” reference measurement can be made for each assay. Thepresent inventors have found however that separate reference zones canbe dispensed with and instead a single shared reference zone willsuffice.

The light source is conveniently an LED. A plurality of LEDs may beemployed. In an embodiment each zone in the assay (detection, referenceor control zone) is illuminated by a respective LED. The one or morephotodetectors may conveniently comprise a photodiode. In a preferredembodiment a single photodiode or other photodetector is employed. Inone embodiment there are four LEDs and a single photodiode.

The assay device may further comprise a control zone which may be asingle control zone provided as part of either the first or secondassay. Alternatively the control zone may be provided on a subsidiaryflow-path to the first and second assay. Provision of a single controlzone reduces further the number of light sources that are needed. Thepurpose of the control zone is to indicate that the assay has beencarried out correctly, namely that fluid sample has been applied to thedevice and that labelled binding reagent has moved along the flow-pathto some extent. The control zone may be provided downstream from thedetection zone. A suitable control zone is disclosed in EP291194 and maycomprise an immobilised binding reagent for a labelled binding reagent.A separate population of labelled binding reagent may be providedupstream from the detection and control zones wherein said separatepopulation of labelled binding reagent is capable of being immobilisedat the control zone but does not become immobilised at the detectionzone in the presence or absence of analyte. The control zone istypically provided downstream from the detection zone. The signalobtained at the control zone may also be referenced with respect to thesignal obtained at the reference zone.

Thus measurement of the signal at the control zone provides a value orindication that the test has been carried out correctly (or incorrectly)for that assay. If for example, the control zone indicates that the testhas been carried out correctly for one assay, an assumption is made thatthe test has been carried out correctly at the other assay. Hence thecontrol zone may be thought of as being “shared” between the assays ofthe assay device. As in the case of a shared reference zone, provisionof a single or “shared” control zone enables a reduced number of opticalcomponents to be used in the device. The rationale for making thisassumption is that it is highly likely that if liquid sample has beenapplied to one assay flow-path, that liquid sample has been applied tothe other flow-path, especially so if the two assay flow-paths areconnected, for example by a common sample receiving means such as aporous sample receiver. Furthermore, if the assay device has for examplebeen subjected to conditions, such as ingress of moisture which may forexample result in poor resuspension of the mobilisable reagents, or hightemperature which may denature the binding reagents, it is likely thatboth flow-paths will be affected. As in the case of a shared referencezone, the concept of a shared control is also counter-intuitive. Thesignal at the control zone is calculated with respect to the signal atthe reference zone.

The reference and control zones may be provided as part of the sameassay or provided as part of different assays. In an exemplaryembodiment, the shared reference and control zones are each provided inseparate assays, e.g. one assay comprises a detection zone and areference zone, and the other assay comprises a detection zone and acontrol zone.

According to a third aspect the invention provides an assay device fordetermining the presence and/or extent of one or more analytes in aliquid sample comprising:

a) first and second assays each comprising a flow-path having adetection zone for immobilising a labelled binding reagent, whereindetection of a labelled binding reagent at one or both detection zonesis indicative of the presence and/or extent of one or more analytes;

b) a shared control zone;

c) one or more light sources to illuminate the detection zones and thecontrol zone;

d) one or more photodetectors to detect light from the detection zonesand the control zone, which photodetector/s generate a signal, themagnitude of which signal is related to the amount of light detected;and

e) signal processing means for processing signals from thephotodetector/s.

According to a fourth aspect, the invention provides an assay reader forreading the result of first and second assays each comprising:

a flow-path, each flow-path comprising a detection zone for immobilisinglabelled binding reagent, wherein detection of labelled binding reagentat one or both detection zones is indicative of the presence and/orextent of one or more analytes; and

a shared control zone;

said assay reader comprising:

-   -   a) one or more light sources to illuminate the detection zones,        and the shared control zone;    -   b) a stored control signal threshold    -   c) one or more photodetector/s to detect light from the        detection zones and the control zone, which photodetector/s        generate a control signal and detection signals, the magnitude        of which signals are related to the amount of light detected;        and    -   d) signal processing means to process signals from the        photodetector/s and to compare the signal obtained from the        control zone to the control signal threshold, and to determine        that both assays have been carried out correctly if the control        signal is equal to or greater than the control signal threshold.

The assay device and reader according to the first, second and thirdaspects of the invention may comprise a control signal threshold.

The control signal threshold may be stored in the device or reader. Thesignal measured from the control zone may be compared to the controlsignal threshold to determine whether sufficient labelled bindingreagent has become immobilised at said zone. If the value of the controlsignal is equal to or exceeds the control signal threshold, the deviceor reader may determine that the assay has been carried outsatisfactorily. If the control signal is less than the control signalthreshold, the device or reader may determine that the assay has beennot been carried out satisfactorily and will provide an error message.

The signal detected from the control zone may be referenced to a signalobtained from a reference zone.

The assay device according to the third aspect may also comprise ashared reference zone.

The first and/or second assay may conveniently comprise a bindingreagent for an analyte or a labelled binding reagent provided in animmobilised form at the detection zone.

By employing a shared reference and/or a shared control zone, the assaydevice of the invention provides for reduced number of zones that needto be interrogated and consequently the number of optical componentsthat need to be employed. The use of shared zones is most effective whenthe assay architectures of the first and second assays are very similaror, if the reference and/or control zone is provided on one or moresubsidiary flow-paths, when the assay architecture of the one or moresubsidiary flow-paths is similar to the first and second assays. Thus,for example, the assays will typically both comprise porous carriers ofsimilar material (e.g. both comprise nitrocellulose carriers). It isalso advantageous to use the same liquid sample for each assay. This maybe conveniently achieved by providing a common sample application regionthat is in fluid communication with both assays. Thus a single liquidsample applied to the device via the common sample application regionmay flow through both the first and second assays. In cases where thefirst and second assays are non-identical, it may be acceptable toprovide a shared reference zone as long as background levels of lightthat would be detected at each assay are sufficiently similar to eachother.

The signal processing means may comprise a central processor unit whichis able to process the signals obtained from the photodetectors from therespective zones and calculate values obtained at the test zone withrespect to the reference zone. The measurement data may be taken atvarious times during the assay and may be taken after the device hasbeen switched on but before fluid sample has been applied to the device,in order to obtain optical values of light transmission or reflectancein the dry state.

An absorbent “sink” can be provided at the distal end of the assayflow-paths. A common sink may be provided or a sink may be provided atthe distal end of each assay. The absorbent sink may preferably comprisea highly absorbent material such as, for example, CF7 Whatman paper, andshould provide sufficient absorptive capacity to remove any unboundconjugate from the vicinity of the detection zones, the reference zoneand the control zone. As an alternative to such a sink it can besufficient to have a length of porous solid phase material which extendsbeyond the detection zone. An advantage of providing a highly absorbentsink is that it removes or substantially removes excess labelled bindingreagent from the flow-paths of the respective assays. This has theeffect of minimising the extent of unbound labelled binding reagent inthe vicinity of respective zones and therefore enables assay flow pathsto be employed in the device that may have differing amounts of labelledbinding reagent.

As an alternative to providing an immobilised binding reagent at thedetection zone, the binding reagent may be provided in a mobilisableform which is capable of binding to an analyte-labelled binding reagentcomplex. The binding reagent may for example be conjugated to a largeparticle such as agarose and the detection zone may comprise a filterwhose pore-size has dimensions smaller than the large particle, butlarger than the size of the labelled binding reagent, such that thefilter is able to trap any labelled binding reagent/analyte/bindingreagent complex present, any labelled binding reagent that is notcomplexed to the capture reagent being able to pass through the filter.Yet alternatively a reagent may be provided in an immobilised form atthe detection zone that is capable of binding a mobilisable labelledbinding reagent/analyte/binding reagent complex. For example the bindingreagent may be provided in a mobilisable form and conjugated to abinding species such as biotin, the reagent immobilised at the detectionzone being a complementary binding partner such as streptavidin.

The assay device may employ a sandwich immunoassay and/or acompetitive/inhibition assay for the determination of an analyte. Anexample of a sandwich immunoassay is where a labelled bindingreagent/analyte/binding reagent complex is formed. The device willtypically comprise a labelled binding reagent for the analyte in amobilisable form provided upstream from a detection zone comprising animmobilised binding reagent for the analyte. Alternatively, inparticular when the analyte of interest is a hapten, the immunoassaydevice may employ a competition reaction wherein a labelled analyte orlabelled analyte analogue competes with analyte present in the samplefor an immobilised binding reagent at a detection zone. The labelledanalyte or labelled analyte analogue may be provided in a mobilisableform upstream from the detection zone. Yet alternatively the assaydevice may employ an inhibition reaction wherein an immobilised analyteor analyte analogue is provided at detection zone, the assay devicecomprising a mobilisable labelled binding reagent for the analyte.

The term “flow-path” for the purposes of this invention refers to asubstrate that is able to convey a liquid from a first position to asecond position and may be for example a capillary channel, amicrofluidic pathway, or a porous carrier such as a lateral flow porouscarrier. The porous carrier may comprise one or a plurality of porouscarrier materials which may overlap in a linear or stacked arrangementor which are fluidically connected. The porous carrier materials may bethe same or different. The first and second assays may be provided onseparate substrates or they may be provided on a common substrate suchthat liquid being conveyed along a flow-path of the first assay is notable to cross over to the flow-path of the second assay. For example,the first and second assays may be provided on the same porous carriersuch that the first and second flow-paths are isolated from each other.This may be achieved for example by laser cutting parts of the porouscarrier to make it non-porous, thus separating the first and secondassays. Alternatively, a non-porous blocking material may be appliedalong a strip to provide two (typically essentially parallel) flow pathson the same porous carrier.

In particular the flow-path may be a lateral flow porous carrier.Suitable materials that may be employed as a porous carrier includenitrocellulose, acetate fibre, cellulose or cellulose derivatives,polyester, polyolefin or glass fibre. The porous carrier may comprisenitrocellulose. This has the advantage that a binding reagent can beimmobilised firmly without prior chemical treatment. If the porous solidphase material comprises paper, for example, the immobilisation of theantibody in the second zone needs to be performed by chemical couplingusing, for example, CNBr, carbonyldiimidazole, or tresyl chloride.

The term “binding reagent” refers to a member of a binding pair, i.e.,two different molecules wherein one of the molecules binds with thesecond molecule through chemical and/or physical means. The twomolecules are related in the sense that their binding with each other issuch that they are capable of distinguishing their binding partner fromother assay constituents having similar characteristics. The members ofthe binding pair are referred to as ligand and receptor (antiligand), abinding pair member and binding pair partner, and the like. A moleculemay also be a binding pair member for an aggregation of molecules; forexample an antibody raised against an immune complex of a secondantibody and its corresponding antigen may be considered to be anbinding pair member for the immune complex.

In addition to antigen and antibody binding pair members, other bindingpairs include, as examples without limitation, biotin and avidin,carbohydrates and lectins, complementary nucleotide sequences,complementary peptide sequences, effector and receptor molecules, enzymecofactors and enzymes, enzyme inhibitors and enzymes, a peptide sequenceand an antibody specific for the sequence or the entire protein,polymeric acids and bases, dyes and protein binders, peptides andspecific protein binders (e.g., ribonuclease, S-peptide and ribonucleaseS-protein), and the like. Furthermore, specific binding pairs caninclude members that are analogues of the original specific bindingmember.

“Label” when used in the context of a labelled binding reagent, refersto any substance which is capable of producing a signal that isdetectable by visual or instrumental means. Various labels suitable foruse in the present invention include labels which produce signalsthrough either chemical or physical means, such as being opticallydetectable. Such labels include enzymes and substrates, chromogens,catalysts, fluorescent compounds, chemiluminescent compounds,electroactive species, dye molecules, radioactive labels and particlelabels. The analyte itself may be inherently capable of producing adetectable signal. The label may be covalently attached to the bindingreagent. In particular the label may be chosen from one that isoptically detectable.

The label may comprise a particle such as gold, silver, colloidalnon-metallic particles such as selenium or tellurium, dyed or colouredparticles such as a polymer particle incorporating a dye, or a dye sol.The dye may be of any suitable colour, for example blue. The dye may befluorescent. Dye sols may be prepared from commercially-availablehydrophobic dyestuffs such as Foron Blue SRP (Sandoz) and Resolin BlueBBLS (Bayer). Suitable polymer labels may be chosen from a range ofsynthetic polymers, such as polystyrene, polyvinyltoluene,polystyrene-acrylic acid and polyacrolein. The monomers used arenormally water-insoluble, and are emulsified in aqueous surfactant sothat monomer micelles are formed, which are then induced to polymeriseby the addition of initiator to the emulsion. Substantially sphericalpolymer particles are produced. An ideal size range for such polymerparticles is from about 0.05 μm to about 0.5 μm. According to anexemplary embodiment the label is a blue polymeric particle.

The dried binding reagents may be provided on a porous carrier materialprovided upstream from a porous carrier material comprising thedetection zone. The upstream porous carrier material may be macroporous.The macroporous carrier material should be low or non-protein-binding,or should be easily blockable by means of reagents such as BSA or PVA,to minimise non-specific binding and to facilitate free movement of thelabelled reagent after the macroporous body has become moistened withthe liquid sample. The macroporous carrier material can be pre-treatedwith a surface active agent or solvent, if necessary, to render it morehydrophilic and to promote rapid uptake of the liquid sample. Suitablematerials for a macroporous carrier include plastics materials such aspolyethylene and polypropylene, or other materials such as paper orglass-fibre. In the case that the labelled binding reagent is labelledwith a detectable particle, the macroporous body may have a pore size atleast ten times greater than the maximum particle size of the particlelabel. Larger pore sizes give better release of the labelled reagent. Asan alternative to a macroporous carrier, the labelled binding reagentmay be provided on a non-porous substrate provided upstream from thedetection zone, said non-porous substrate forming part of the flow-path.

The porous carrier may comprise a glass-fibre macroporous carrierprovided upstream from and overlapping at its distal end anitrocellulose porous carrier.

The liquid sample can be derived from any source, such as an industrial,environmental, agricultural, or biological source. The sample may bederived from or consist of a physiological source including blood,serum, plasma, interstitial fluid, saliva, sputum, ocular lens liquid,sweat, urine, milk, mucous, synovial liquid, peritoneal liquid,transdermal exudates, pharyngeal exudates, bronchoalveolar lavage,tracheal aspirations, cerebrospinal liquid, semen, cervical mucus,vaginal or urethral secretions and amniotic liquid. In particular thesource may be human and in particular the sample may be urine.

“Light” as used herein is intended to encompass any suitableelectromagnetic radiation, regardless of wavelength. Notwithstandingthis, the invention is primarily intended to utilise light in thevisible part of the spectrum, and “light source” and “photodetector”should be construed accordingly as encompassing respectively any sourceof, and means for detecting, electromagnetic radiation, but especiallyrelating to radiation of visible wavelengths (i.e. in the range of about390-800 nm).

The photodetector/s will detect light from one or more zones of theassay device. The light may actually originate from those zones, forexample, if the label is fluorescent or the like. More normally however,the photodetector/s will detect light which appears to emanate fromthose zones i.e., light which originates from the light source and isreflected and/or transmitted by the zone onto the photodetector.

Analytes include, but are not limited to, toxins, organic compounds,proteins, peptides, micro-organisms, bacteria, viruses, amino acids,nucleic acids, carbohydrates, hormones, steroids, vitamins, drugs(including those administered for therapeutic purposes as well as thoseadministered for illicit purposes), pollutants, pesticides, andmetabolites of or antibodies to any of the above substances. The termanalyte also includes any antigenic substances, haptens, antibodies,macromolecules, and combinations thereof.

The assay device may determine one or more analytes.

The assay device may be capable of determining the amount or presence ofan analyte over an extended analyte range, wherein the first assay iscapable of determining the level of analyte at a lower concentrationrange and the second assay is capable of determining the level ofanalyte in a liquid sample at a higher concentration range.

There are several ways in which an assay may be prepared in order tomeasure analyte at a higher analyte range.

For example, the assay device may comprise a scavenger assay comprisinga labelled binding reagent for the analyte and a scavenger bindingreagent for the analyte, provided upstream from the detection zone. Thescavenger binding reagent serves to remove excess analyte and lower thesensitivity of the assay. This has the effect of increasing the dynamicrange of the assay enabling measurement at higher analyte levels. Thescavenger binding reagent may be may be immobilised, mobilisable orboth. The scavenger binding reagent may be provided at either the sameregion of the porous carrier as the mobilisable labelled bindingreagent, upstream from it or downstream from it. The scavenger bindingreagent may bind to the same binding region of the analyte as themobilisable labelled binding reagent or to a different region of theanalyte than the labelled binding reagent. The scavenger reagent mayhave a different affinity for the analyte than the mobilisable labelledbinding reagent of the second assay. In an exemplary embodiment, thescavenger binding reagent has a higher affinity for the analyte than themobilisable binding reagent of the second assay. The amount of scavengerbinding reagent may be varied to change the sensitivity of the assay toanalyte concentration. Increasing the amount of scavenger bindingreagent present lowers the sensitivity of the assay due to the fact thatthe scavenger binding reagent is able to bind more analyte, effectivelylowering the proportion of labelled binding reagent that is able to bindto the detection zone.

In order to increase the dynamic range of the assay, the assay devicemay for example comprise multiple detection zones, wherein eachdetection zone is capable of binding analyte at different analyteconcentration levels. For example the respective zones may comprisebinding reagent for the analyte having a differing affinities for theanalyte.

Other ways to increase the dynamic range of the assay are to provide anassay device comprising a sandwich binding assay and a competition orinhibition assay. For example, the sandwich assay may be the highsensitivity assay, namely it is capable of measuring analyte at a lowerconcentration range and the inhibition or competition assay may be a lowsensitivity assay, namely it is capable of measuring analyte at a higherconcentration range. A further way is to alter the affinity or amount ofthe labelled binding reagent or the immobilised reagent at the detectionzone. A high affinity binding reagent will have a higher analytesensitivity than a lower affinity binding reagent. Similarly a lowconcentration of binding reagent will have a lower analyte sensitivitythan a high concentration of binding reagent. The assay sensitivity canbe changed by altering the ratio of binding reagent to the label of thelabelled binding reagent. If a particle is used as the label, then thequantity of the binding reagent applied to the label can be altered toalter assay sensitivity. A further way to manipulate the sensitivity ofan assay is to vary the quantity of the label used in the assay. Forexample the sensitivity of an assay may be lowered by reducing the ratioof binding reagent to labelled species for the labelled binding reagent.

A further means of manipulating the sensitivity of an assay is to alterthe optical density of a label. The assay sensitivity can be lowered byuse of a label with a low optical density. This may be achieved forexample by provision of a polymer particle label having a lowconcentration of dye or by use a coloured label which is less sensitiveto an optical detector.

Yet a further way to measure high analyte levels is to employ anon-particulate labelled binding reagent. High levels of analyte whenmeasured by way of a sandwich binding assay require high levels ofbinding reagent. In the case wherein the label is a particle label,provision of high levels of analyte within or on the porous carrier cangive rise to steric hindrance resulting in poor assay sensitivity.Conversely, at lower analyte levels, the use of a non-particle labelledbinding reagent can give rise to a low signal due to the low opticaldensity. However, at high analyte levels, non-particle labels may bepresent at sufficiently high levels to be readily detected. An exampleof a optically detectable non-particulate label may be a dye. The dyemay be fluorescent.

Assay sensitivity may be influenced by the flow rate of the porouscarrier. A way to lower the sensitivity of the assay is to employ aporous carrier (such as nitrocellulose) having a higher flow rate.

The sensitivity of an assay may be further manipulated by modifying therate at which the labelled binding reagent is released from its origin.A further way to lower analyte sensitivity is to provide for a rapidrelease of the labelled binding reagent from the porous carrier duringcontact with the liquid sample. The release of the labelled bindingreagent can be modified by the provision of sugars, proteins or otherpolymeric substances such as methylcellulose within the device.

According to a particular embodiment, the assay device comprises ascavenger assay comprising a mobilisable second (scavenger) bindingreagent for the analyte and a mobilisable binding reagent for theanalyte provided upstream from the detection zone.

According to a particular embodiment the first assay is capable ofmeasuring analyte in a lower analyte concentration range and the secondassay is capable of measuring analyte in a higher analyte concentrationrange. The first assay may comprise a shared reference zone and thesecond assay may comprise a shared control zone.

The first assay may comprise a labelled binding reagent providedupstream from a detection zone and the second assay may comprise alabelled binding reagent and a mobilisable scavenger binding reagentprovided upstream from a detection zone. The scavenger binding reagentmay be provided at the same position or in the region of the labelledbinding reagent upstream from the detection zone.

In particular the analyte to be determined is hCG and the liquid sampleis urine.

In order to measure an analyte concentration over a certain range it isimportant to ensure that there is sufficient labelled binding reagentpresent such that the assay signal does not become saturated.Measurement of large amounts of analyte often requires a correspondingincrease in the amount of labelled binding reagent to avoid theso-called “hook effect” or saturation of the assay signal withincreasing analyte concentration. Variation in the control signal hasbeen shown to occur particularly in the case where there is an increasedamount of binding reagent present.

Where first and second assays are provided having differing amounts oflabelled binding reagent, it has been shown to be advantageous toprovide the reference zone as part of the assay having a lower level oflabelled binding reagent.

According to an embodiment, the assay device is capable of measuringanalyte at a higher analyte range. There are several ways of providingsuch a device.

For example, the assay device may comprise a labelled binding reagentfor the analyte and a second binding reagent for the analyte, providedupstream from the detection zone. The second binding reagent serves toremove excess analyte and lower the sensitivity of the assay. This hasthe effect of increasing the dynamic range of the assay enablingmeasurement at higher analyte levels. The second binding reagent may bemay be immobilised, mobilisable or both. The second binding reagent maybe provided at either the same region of the porous carrier as themobilisable labelled binding reagent, upstream from it or downstreamfrom it. The second binding reagent may bind to the same binding regionof the analyte as the mobilisable labelled binding reagent or to adifferent region of the analyte than the labelled binding reagent. Thesecond reagent may have a different affinity for the analyte than themobilisable labelled binding reagent of the second assay. In anexemplary embodiment, the second binding reagent has a higher affinityfor the analyte than the mobilisable binding reagent of the secondassay. The amount of second binding reagent may be varied to change thesensitivity of the assay to analyte concentration. Increasing the amountof second binding reagent present lowers the sensitivity of the assaydue to the fact that the second binding reagent is able to bind moreanalyte, effectively lowering the proportion of labelled binding reagentthat is able to bind to the detection zone.

In order to increase the dynamic range of the assay, the assay devicemay for example comprise multiple detection zones, wherein eachdetection zone is capable of binding analyte at different analyteconcentration levels. For example the respective zones may comprisebinding reagent for the analyte having a differing affinities for theanalyte.

Other ways to increase the dynamic range of the assay are to provide anassay device comprising a sandwich binding assay and a competition orinhibition assay. For example, the sandwich assay may be the highsensitivity assay, namely it is capable of measuring analyte at a lowerconcentration range and the inhibition or competition assay may be a lowsensitivity assay, namely it is capable of measuring analyte at a higherconcentration range. A further way is to alter the affinity or amount ofthe labelled binding reagent or the immobilised reagent at the detectionzone. A high affinity binding reagent will have a higher analytesensitivity than a lower affinity binding reagent. Similarly a lowconcentration of binding reagent will have a lower analyte sensitivitythan a high concentration of binding reagent. The assay sensitivity canbe changed by altering the ratio of binding reagent to the label of thelabelled binding reagent. If a particle is used as the label, then thequantity of the binding reagent applied to the label can be altered toalter assay sensitivity. A further way to manipulate the sensitivity ofan assay is to vary the quantity of the label used in the assay. Forexample the sensitivity of an assay may be lowered by reducing the ratioof binding reagent to labelled species for the labelled binding reagent.

A further means of manipulating the sensitivity of an assay is to alterthe optical density of a label. The assay sensitivity can be lowered byuse of a label with a low optical density. This may be achieved forexample by provision of a polymer particle label having a lowconcentration of dye or by use a coloured label which is less sensitiveto an optical detector.

Yet a further way to measure high analyte levels is to employ anon-particulate labelled binding reagent. High levels of analyte whenmeasured by way of a sandwich binding assay require high levels ofbinding reagent. In the case wherein the label is a particle label,provision of high levels of analyte within or on the porous carrier cangive rise to steric hindrance resulting in poor assay sensitivity.Conversely, at lower analyte levels, the use of a non-particle labelledbinding reagent can give rise to a low signal due to the low opticaldensity. However, at high analyte levels, non-particle labels may bepresent at sufficiently high levels to be readily detected. An exampleof a optically detectable non-particulate label may be a dye. The dyemay be fluorescent.

Assay sensitivity may be influenced by the flow rate of the porouscarrier. A way to lower the sensitivity of the assay is to employ aporous carrier (such as nitrocellulose) having a higher flow rate.

The sensitivity of an assay may be further manipulated by modifying therate at which the labelled binding reagent is released from its origin.A further way to lower analyte sensitivity is to provide for a rapidrelease of the labelled binding reagent from the porous carrier duringcontact with the liquid sample. The release of the labelled bindingreagent can be modified by the provision of sugars, proteins or otherpolymeric substances such as methylcellulose within the device.

According to a particular embodiment, the assay device comprises amobilisable second binding reagent for the analyte and a mobilisablebinding reagent for the analyte provided upstream from the detectionzone. The second binding reagent may be provided at the same or similarposition upstream from the detection zone as the labelled bindingreagent.

According to a particular embodiment, the assay device comprises twoassays each comprising an flow-path, wherein the first assay is capableof measuring analyte in a lower analyte concentration range and thesecond assay is capable of measuring analyte in a higher analyteconcentration range. The first assay may comprise a shared referencezone and the second assay may comprise a shared control zone.

The assay device of the invention may be used to measure the extent orpresence of hCG over an extended concentration range. The range may varyfrom between about 10 mIU to about 250,000 mIU.

The second assay may comprise a labelled binding reagent for the analyteand a second binding reagent for the analyte. The first assay maycomprise labelled binding reagent for the analyte provided upstream fromthe detection zone.

The assay device may comprise one or more further measurement thresholdvalues to indicate the level of analyte in a certain analyte range. Inan embodiment, the assay device comprises a first and second measurementthresholds, wherein an analyte measurement signal of less than the firstmeasurement threshold is indicative of the absence of analyte or theabsence of analyte above a certain level and wherein an analytemeasurement signal greater than the second threshold is indicative ofthe level of analyte in a second concentration range and a measurementsignal of less than the second threshold is indicative of the level ofanalyte in a first concentration range. According to a particularembodiment, the assay device additionally comprises a third measurementthreshold, wherein an analyte measurement signal greater than the thirdthreshold is indicative of the level of analyte in a third concentrationrange.

In particular the assay device may be capable of measuring the presenceand extent of the analyte hCG analyte in a liquid sample, in particularurine, of a female mammalian subject. The assay device may comprise afirst measurement threshold, wherein hCG analyte signal levels of belowthe threshold are indicative or being not pregnant and wherein hCGanalyte signal levels greater than or equal to the first measurementthreshold are indicative of being pregnant, wherein the device comprisesat least a further measurement threshold. In addition the assay devicemay provide an indication of the extent of pregnancy. The assay devicemay provide a time-based indication to the user, such as the extent ofpregnancy in units of days or weeks.

A typical full assay development time for an assay test for thedetermination of hCG in urine is 3 minutes.

It is an desirable object of the invention to reduce the number ofoptical components, this may be conveniently achieved, where thereference zone is provided as part of one assay and the control zone isprovided as part of the other assay. According to an embodiment, thereference zone is provided as part of a first assay having a lower levelof labelled binding reagent and the control zone is provided as part ofa second assay having a higher level of binding reagent.

According to an embodiment, the assay device comprises four lightsources, wherein the light sources are arranged to illuminate thedetection zones of the first and second assay and the shared control andreference zones, each zone being illuminated by a respective lightsource. One or more photodetectors may be positioned to detect reflectedand/or transmitted light from the respective zones. According to anembodiment, a single photodetector may be employed to detect light fromall of the zones. This may be achieved by, for example, illuminating therespective zones sequentially such that the device is able to recognisefrom which zone light detected at the photodetector is emanating. Thesequential illumination process may be repeated with a fixed or variedfrequency during the duration of the assay such that the levels ofsignal over time at each zone may be monitored. In addition the changein levels of light detected from one or more zones may be used todetermine whether and when a fluid sample has been applied to the deviceand to determine the flow-rate of liquid sample along the device.Determination of the flow-rate may be used as a further quality controlcheck, for example the assay may be rejected if the flow-rate is eithergreater than or less than set levels. A suitable flow-rate detectionmethod and means is disclosed by EP1484641.

The labelled binding reagent typically accumulates at the detection zoneover a period of time for a sandwich immunoassay and thus the rate ofincrease of signal over time may be monitored. The device may determinethe result after the signal has reached equilibrium or more typicallybefore the reaction has reached equilibrium. The device may provide aquantitative result such as a individual value, a semi-quantitativeresult or range such as 1-10, 11-20 and so on, or a qualitative resultsuch as YES/NO. The device may determine the result with respect to oneor more signal thresholds. The device may have a fixed measurement timeor provide an early result before the fixed measurement time haselapsed. An early result may for example be given in the case where thedevice determines that the signal level will never exceed a particularthreshold or exceeds a particular threshold at an early stage. In theseparticular cases the device may call an early NO or YES measurement,indicating the absence or presence of analyte with respect to aparticular base level (which may be zero). An assay device employing anearly result determination method is disclosed by EP1464613.

The assay device may be used to determine whether a subject is pregnantor not (namely whether the liquid sample contains hCG above a certainlevel) and may also employ further thresholds indicating to the user theextent of pregnancy. The extent of pregnancy may be displayed in termsof a time-based or concentration-based measurement.

The assay device will typically comprise a housing. The housing may befluid impermeable and constructed from a suitable plastics material,such as ABS. The assay device may further comprise a sample receivingmember for receiving the fluid sample. The sample receiving member mayextend from the housing.

The housing may be constructed of a fluid impermeable material. Thehousing will also desirably exclude ambient light. The housing or casingwill be considered to substantially exclude ambient light if less than10%, preferably less than 5%, and most preferably less than 1%, of thevisible light incident upon the exterior of the device penetrates to theinterior of the device. A light-impermeable synthetic plastics materialsuch as polycarbonate, ABS, polystyrene, polystyrol, high densitypolyethylene, or polypropylene containing an appropriate light-blockingpigment is a suitable choice for use in fabrication of the housing. Anaperture may be provided on the exterior of the housing whichcommunicates with the assay provided within the interior space withinthe housing. Alternatively the aperture may serve to allow a poroussample receiver to extend from the housing to a position external fromthe housing.

Also provided within the housing will typically be a power source. Thedevice will typically comprise a display means to display the result ofthe assay as well as a memory means to store data. Conveniently thedisplay means comprises an LCD.

The display means may further display further information such as anerror message, personal details, time, date, and a timer to inform theuser how long the assay has been measured for. The information displayedby the assay may be indicated in words, numbers or symbols, in any font,alphabet or language, for example, “positive”, “negative”, “+”, “−”,“pregnant”, “not pregnant”, “see your doctor”, “repeat the test”.

The assay device may comprise a porous sample receiver in fluidconnection with and upstream from the flow-path. The assay device maycomprise more than one assay flow-path each comprising a detection zone,in which case a single porous sample receiver may be provided which iscommon to the multiple assay flow paths. Thus a fluid sample applied tothe porous sample receiver of the device is able to travel along theflow-paths of the respective assays to the respective detection zones.The porous sample receiver may be provided within a housing or may atleast partially extend out of said housing and may serve for example tocollect a urine stream. The porous sample receiver may act as a fluidreservoir. The porous sample receiving member can be made from anybibulous, porous or fibrous material capable of absorbing liquidrapidly. The porosity of the material can be unidirectional (i.e. withpores or fibres running wholly or predominantly parallel to an axis ofthe member) or multidirectional (omnidirectional, so that the member hasan amorphous sponge-like structure). Porous plastics material, such aspolypropylene, polyethylene (preferably of very high molecular weight),polyvinylidene fluoride, ethylene vinylacetate, acrylonitrile andpolytetrafluoro-ethylene can be used. Other suitable materials includeglass-fibre. Provision of a common porous sample receiver enables asingle sample to be provided simultaneously to the flow-paths of thefirst and second assays and further increases the effectiveness ofproviding a shared reference and/or shared control zone.

In a fifth aspect, the invention provides a method of performing anassay to determine the presence and/or extent of one or more analytes,the method comprising the step of contacting a liquid sample with anassay device in accordance with the first and third aspects of theinvention.

OVERVIEW OF THE FIGURES

FIG. 1 is a view of an assay device in accordance with the invention;

FIG. 2 is a schematic view of the assay flow-paths according to anexemplary embodiment in accordance with the invention;

FIG. 3 is a view of the arrangement of light sources and photodetectorof the embodiment shown in FIG. 2;

FIG. 4 is a schematic cross-sectional view of part of one embodiment ofthe assay device illustrating the relative positions of some of theassay components;

FIGS. 5a and 5b are views of the underside of a baffle arrangement alsoshowing some of the optical components of the embodiment shown in FIG.3; and

FIG. 6 is a top view of the part of the assay device embodiment depictedin preceding figures, and illustrating a lateral flow test-strip in situin the assay device.

DETAILED DESCRIPTION

An external top view of an assay device is shown in FIG. 1. The device(10) is elongate having a length of about 14 cm and a width of about 25mm. The casing (11) may be formed of a suitable liquid impermeablecasing such as polycarbonate, ABS, polystyrene, high densitypolyethylene, or polypropylene. The external porous sample receiver (12)may be formed of any bibulous, porous or fibrous material capable ofabsorbing liquid rapidly. Also shown is an LCD display (15) fordisplaying the results of the assay. Also provided within the assaydevice and not shown, are the assay flow-paths, light sources,photodetector, a power source and associated electronic components.

FIG. 2 shows the layout of the photodetector and the individual assayporous carriers of an assay device according to an exemplary embodiment.Assay device (20) has a common sample application region (21) whichfluidically connects first and second assays (22) and (23). A singlephotodetector (4) is provided between the two assays to detect lightfrom the respective zones. Zones (24) and (25) correspond respectivelyto a detection and control zone on first assay (22). Zones (26) and (27)correspond respectively to a detection and reference zone on secondassay (23). Not shown are the corresponding four LEDs which eachilluminate a respective zone through appropriately positioned windows.

FIG. 3 shows a view of the arrangement according to an exemplaryembodiment comprising a single photodetector (32) and four LEDs (31).The active area of the photodetector is 1.5 mm×1.5 mm.

FIG. 4 shows a cross-sectional schematic view of the assay device (40)showing the relative positions of some of the components. Light from LED(41) illuminates a zone of strip (42) and light reflected from the zoneis detected by the photodetector (44). Similarly, light from LED (45)illuminates a zone of strip (46) and reflected light is detected by thephotodetector. Provided are dividers (47) which prevent light from theLED being directly incident on the photodetector. Also provided is asloping member (48) which serves to prevent illumination of strip (46)by LED (41) and correspondingly the illumination of strip (42) by LED(45) whilst allowing light reflected from the respective test strips tobe detected by the photodetector. The sloping member also serves toguide reflected light from the test-strips onto the photodetector. TheLEDs are mounted on a surface (49) made from printed circuit board.

FIG. 5a illustrates an underside view of the baffle arrangement of theexemplified embodiment. Light from the LEDs, of which one (denoted byreference numeral 51) is shown, illuminates a zone of an assay strip(not shown) through an aperture. Each LED is associated with arespective aperture. In the Figure, an exemplary aperture is denoted byreference numeral 55. Light is reflected from the strip ontophotodetector (52). Also shown is the sloping member (53) and divider(56). Adjacent LEDs are screened from one another by baffles (54).

FIG. 5b shows an underside view of the baffle arrangement of theexemplified embodiment from a different perspective. The sloping member(53) is symmetrical about axis (57) and serves to guide reflected lightfrom all four LEDs (not shown) onto the photodetector (not shown).

FIG. 6 shows top view of the assay device looking down onto a test-strip(61) located over the apertures (62) and held in position by locatingpins (63). The LEDs and photodetector can be partially seen through theapertures.

Example 1

The value of the signal determined from the respective zones for anassay device comprising a low sensitivity test-zone (for measuring highanalyte concentration) and a high sensitivity test-zone (for measuringlow analyte concentration) is determined by the signal computation meansas follows:

The use of the strips and windows are defined in the table below (seeFIG. 2)

Strip Window 1 Window 2 A Low Sensitivity test line (LS) Control line(Ctrl) B High Sensitivity test line (HS) Reference window (Ref)

Measurements of the light reflected from each window are takenapproximately twice a second and a low pass digital filter is used toreject noise and smooth the data. Filtered values are used for detectingflow and determining the result and are expressed in terms of normalisedpercentage relative attenuation (% A). This takes into account andminimises any variations in the optical components both within thedevice and between devices.

The measured value is inversely proportional to the quantity of lightreflected.

For each window, the window ratio at the reference, control, and testwindows is equal to the measured value when the porous carrier is dry,t=0 (prior to addition of sample), divided by the measured value at timet after addition of sample:

Calculation of Filtered Window Ratios

For each time point t the window ratios for each window are evaluated asfollows:

$\begin{matrix}{{{Ref}\mspace{11mu} {ratio}_{t}} = \frac{{filtered}\mspace{14mu} {reference}\mspace{14mu} {window}\mspace{14mu} {value}_{{time} = 0}}{{filtered}\mspace{14mu} {reference}\mspace{14mu} {window}\mspace{14mu} {value}_{{time} = t}}} \\{{{HS}\mspace{14mu} {ratio}_{t}} = \frac{{filtered}\mspace{14mu} {HS}\mspace{14mu} {test}\mspace{14mu} {window}\mspace{14mu} {value}_{{time} = 0}}{{filtered}\mspace{14mu} {HS}\mspace{14mu} {test}\mspace{14mu} {window}\mspace{14mu} {value}_{{time} = t}}} \\{{{LS}\mspace{14mu} {ratio}_{t}} = \frac{{filtered}\mspace{14mu} {LS}\mspace{14mu} {test}\mspace{14mu} {window}\mspace{14mu} {value}_{{time} = 0}}{{filtered}\mspace{14mu} {LS}\mspace{14mu} {test}\mspace{14mu} {window}\mspace{14mu} {value}_{{time} = t}}} \\{{{Ctrl}\mspace{11mu} {ratio}_{t}} = \frac{{filtered}\mspace{14mu} {Ctrl}\mspace{14mu} {window}\mspace{14mu} {value}_{{time} = 0}}{{filtered}\mspace{14mu} {Ctrl}\mspace{14mu} {window}\mspace{14mu} {value}_{{time} = t}}}\end{matrix}$

Calculation of Filtered % A Values

The normalised percentage relative attenuation (% A) is given by thedifference of the reference (ref.) window ratio and the window ratiobeing considered (control or test windows) divided by the referencewindow ratio and multiplied by 100%.

For each time point t, % A values are calculated for the HS test line,LS test line and control line, wherein:

$\begin{matrix}{{{HS}_{t}\left( {\% \mspace{11mu} A} \right)} = {\frac{{{Ref}\mspace{14mu} {ratio}_{t}} - {{HS}\mspace{14mu} {test}\mspace{14mu} {ratio}_{t}}}{{Ref}\mspace{14mu} {ratio}_{t}} \times 100\%}} \\{{{LS}_{t}\mspace{11mu} \left( {\% \mspace{11mu} A} \right)} = {\frac{{{Ref}\mspace{11mu} {ratio}_{t}} - {{LS}\mspace{14mu} {test}\mspace{14mu} {ratio}_{t}}}{{Ref}\mspace{14mu} {ratio}_{t}} \times 100\%}} \\{{{Ctrl}_{t}\; \left( {\% \mspace{11mu} A} \right)} = {\frac{{{Ref}\mspace{11mu} {ratio}_{t}} - {{Ctrl}\mspace{11mu} {ratio}_{t}}}{{Ref}\; {ratio}_{t}} \times 100\%}}\end{matrix}$

Construction of Assay Devices

An assay device according to the first aspect of the invention wasconstructed comprising a first assay test-strip comprising a labelledbinding reagent provided upstream from a detection zone and a secondassay test-strip comprising a labelled binding reagent and a second(scavenger) binding reagent for the analyte as well as labelled bindingreagent for a control zone provided upstream from a detection zone and acontrol zone.

Preparation of the First Assay Test-Strip

The detection zone was prepared by dispensing a line of anti-β-hCGantibody (in-house clone 3468) at a concentration of 3 mg/ml in PBSAbuffer, at a rate of 1 μl/cm on onto bands of nitrocellulose ofdimensions 350 mm length×40 mm width (Whatman) having a pore-size of 8microns and a thickness between 90-100 microns which had been laminatedto a 175 micron backing layer. The anti-β-hCG antibody was applied usingthe Biodot xyz3050 dispensing platform as a line ˜1.2 mm in width and˜300 mm in length at a position of 10 mm along the length of thenitrocellulose.

The bands of nitrocellulose were dried using Hedinair drying oven serial#17494 set at 55° C. and speed 5 (single pass).

The nitrocellulose was subsequently blocked using a blocking buffercomprising a mixture of 5% ethanol (BDH Analar 104766P) plus 150 mMSodium Chloride (BDH Analar 10241AP) plus 50 mM trizma base from (SigmaT1503) plus Tween 20 (Sigma P1379) and 1% (w/v) polyvinyl alcohol (PVA,Sigma 360627).

The blocking buffer was applied at a rate of 1.750 mm to the proximalend of the band. Once the blocking solution had soaked into the membranea solution of 2% (w/v) sucrose (Sigma S8501 in deionised water) wasapplied using the same apparatus at a rate of 1.6 μl/mm and allowed tosoak into the nitrocellulose membrane for ˜5 minutes).

The bands of NC were then dried using a Hedinair drying oven serial#17494 set at 75° C. and speed 5 (single pass).

Preparation of the Mobilisable Labelled Binding Reagent on the FirstPorous Carrier Material.

Labelled binding reagent was prepared according to the followingprotocol:

Coating Latex Particles with Anti-α hCG

-   1. Dilute blue latex particles from Duke Scientific (400 nm in    diameter, DB1040CB at 10% solids (w/v)) to 2% solids (w/v) with 100    mM di-sodium tetra borate buffer pH 8.5 (BDH AnalaR 102676G) (DTB).-   2. Wash the diluted latex by centrifuging a volume of (2 mls) of    diluted latex in two Eppendorf centrifuge tubes at 17000 rpm (25,848    rcf) for 10 minutes on an Heraeus Biofuge 17RS centrifuge. Remove    and discard the supernatant and re-suspend the pellets in 100 mM DTB    to give 4% solids (w/v) in a total volume of 1 ml.-   3. Prepare a mixture of ethanol and sodium acetate (95% Ethanol BDH    AnalaR 104766P with 5% w/v Sodium Acetate Sigma S-2889).-   4. Add 100 μls ethanol-sodium acetate solution to the washed latex    in step 2 (this is 10% of the volume of latex).-   5. Dilute the stock antibody (in-house clone 3299) to give ˜1200    μg/ml antibody in DTB.-   6. Heat a volume of 1 ml of the diluted antibody from step 5 in a    water bath set at 41.5° C. for ˜2 minutes. Also heat the washed    latex plus ethanol-sodium acetate from step 4 in the same water bath    for 2 minutes.-   7. Add the diluted antibody to the latex plus ethanol-acetate, mix    well and incubate for 1 hour in the water bath set at 41.5° C.    whilst mixing using a magnetic stirrer and a magnetic flea placed in    the mixture.-   8. Prepare 40 mg/ml Bovine Serum Albumin (BSA) Solution (Intergen    W22903 in de-ionised water). Block the latex by adding an equal    volume of 40 mg/ml BSA to the mixture of    latex/antibody/ethanol-acetate and incubate in the water bath at    41.5° C. for 30 minutes with continued stirring.-   9. Centrifuge the mixture at 17000 rpm for 10 minutes as in step 2,    (split the volume into 1 ml lots between Eppendorf tubes). Remove    and discard the supernatant and re-suspend the pellet in 100 mM DTB.    Repeat the centrifugation as in step 2, remove and discard the    supernatant and re-suspend in pellet in Air Brushing Buffer (20%    (w/v) Sucrose Sigma 58501, 10% BSA (w/v) in 100 mM Trizma Base Sigma    T1503 pH to 9). Add Air Brushing Buffer to give 4% solids (w/v)    latex.

The conjugated latex was and sprayed in a mixture of BSA and sucroseonto a glass-fibre porous carrier (F529-09, Whatman) at a rate of 50g/hr and 110 mm/s and dried using a Hedinar Conveyor Oven Serial number17494 set at 65° C. and speed 5 (single pass).

The zone chosen as the reference zone was at a distance of 13 mm alongthe nitrocellulose, namely downstream of the detection zone.

The glass fibre material comprising the labelled binding reagent wasattached to the nitrocellulose membrane using a clear adhesive coatedlaminate film (Ferrisgate, 38 mm wide) arranged such that the labelledreagent was uppermost and the glass fibre overlapped the surface of thenitrocellulose by ˜2 mm along the length (350 mm) of the band ofnitrocellulose membrane. The glass fibre was attached to the end of thenitrocellulose such that it was upstream of the detection zone.

The laminated sheet was subsequently cut into test-strips comprising aglass fibre porous carrier material having a width of 6 mm and a length25 mm, with the labelled reagents having been applied 20 mm along thelength of the glass fibre, provided upstream from and overlapping by 2mm, a nitrocellulose membrane having a width of 6 mm and a length of 40mm.

Preparation of the Second Assay Test-Strip.

The detection zone was prepared as follows:

MAb mouse anti-human β-hCG antibody (clone 3468) 3 mg/ml in PBSA bufferwas plotted at 1 μl/cm onto nitrocellulose (of type and dimensions asthat according to the first assay) at the 10 mm position using a BiodotXYZ3050 dispensing platform to provide a sole detection zone for thefirst assay.

The control zone was prepared as follows:

Goat-anti-Rabbit antibody (Lampire) at 2 mg/ml in PBSA buffer wasplotted at 1 μl/cm onto the same nitrocellulose as used for the secondassay, at the 13 mm position using a Biodot XYZ3050 dispensing platformto provide a sole control zone for the assay device.

Mouse-anti-human α-hCG mAb (clone 3299) conjugated to 400 nm bluepolystyrene latex (Duke Scientific) was mixed with scavenger antibodymAb mouse anti-human β-hCG (in-house clone 3468) at 3 mg/ml to give afinal % blue latex of 3%, a final 3468 concentration of 0.075 mg/ml and0.06 mg/ml concentration of the free anti-β hCG antibody. The resultingmixture was airbrushed onto Whatman glass fibre (F529 25 mm wide reels)using the BIODOT XYZS (serial number 1673) at 90 g/hr sprayed at 2.02μg/cm onto F529-09 glass fibre at approximately the 20 mm position. Thesprayed solution spread out to form a band that was approximately 7 mmin length.

Labelled binding reagent for the control zone was also deposited ontothe same region of the porous carrier as the labelled binding reagentfor the analyte as follows: Rabbit IgG (Dako) was conjugated to 400 nmblue latex polystyrene latex (Duke Scientific) in BSA/sucrose to give afinal % blue latex of 0.7% solids and sprayed at 65 g/hr onto glassfibre.

The glass fibre was dried using a Hedinar Conveyor Oven Serial number17494 set at 65° C. and speed 5 (single pass). A second pass of latexwas deposited onto the glass fibre by repeating the above however at anoffset of ˜0.8 mm from the original position of spray (furtherdownstream of the glass fibre). The glass fibre as dried as describedabove.

The glass fibre material with sprayed latex was attached to thenitrocellulose membrane using a clear adhesive coated laminate film(Ferrisgate, 38 mm wide) arranged such that the sprayed latex wasuppermost and the glass fibre overlapped the surface of thenitrocellulose by approximately 2 mm along the length (350 mm) of theband of nitrocellulose membrane. The glass fibre was provided upstreamfrom the nitrocellulose membrane and the binding reagents were providedtowards the distal end of the glass fibre.

The laminated sheet was subsequently cut into test-strips comprising aglass fibre porous carrier material having a width of 6 mm and a length25 mm, with the labelled reagents having been applied 20 mm along thelength of the glass fibre, provided upstream from and overlapping by 2mm, a nitrocellulose membrane having a width of 6 mm and a length of 40mm. A porous sample receiver (Filtrona) of 45 mm length, 12 mm width anda thickness of approximately 2.5 mm was provided upstream from andoverlapping the first porous carrier material by approximately 3 mm.

Labelled binding reagent for the control zone was also deposited ontothe same region of the porous carrier as the labelled binding reagentfor the analyte as follows:

Rabbit IgG (Dako) was conjugated to 400 nm blue latex polystyrene latex(Duke Scientific) in BSA/sucrose to give a final % blue latex of 0.7%solids and sprayed at 65 g/hr onto glass fibre (F529-09).

The first and second assay test-strips were positioned parallel to oneanother and a common polyester sample application pad (505521, Filtrona)was overlaid at the upstream ends of both assays. A common cottonabsorbent sink pad (CF7, Whatman) was overlaid downstream of thereference and control zones.

The assay device was prepared by mounting the assay strips in a parallelconfiguration into a plastic housing comprising the optical components.The LEDs were arranged such that the four LEDs were positioned in closeproximity to the respective four zones (2 detection zones and thereference and control zones) in an offset position and above the planeof the assays. A single photodetector was positioned between and abovethe plane of the two assays and positioned in the middle of the assaystrips (see FIG. 2).

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An assay device for determining the presence and/or extent of one ormore analytes in a liquid sample comprising: a) a first and secondflow-path, each flow-path having a detection zone for immobilizing alabeled binding reagent, wherein detection of a labeled binding reagentat one or both detection zones is indicative of the presence and/orextent of one or more analytes; b) a common sample application zonewhich fluidically connects the first and second flow-paths; c) a sharedreference zone; d) one or more light sources to illuminate the detectionzones and the shared reference zone; e) one or more photodetectors todetect light from the detection zones and the shared reference zone,which photodetector generates a signal, the magnitude of which signal isrelated to the amount of light detected; and f) signal processing meansfor processing signals from the photodetector.
 2. The assay deviceaccording to claim 1, wherein the first and or second flow-pathscomprise a labeled binding reagent for an analyte or analyte analogueprovided in a mobilizable form upstream from the detection zone in a drystate prior to use of the device.
 3. The assay device according to claim1, wherein the first and/or second flow-path comprises a binding reagentfor an analyte or a labeled binding reagent provided in an immobilizedform at the detection zone.
 4. The assay device according to claim 1,further comprising a shared control zone.
 5. The assay device accordingto claim 1, wherein the shared reference zone is comprised as part ofeither the first or second assay.
 6. The assay device according to claim4, wherein the control zone is comprised as part of either the first orsecond flow-path.
 7. The assay device according to claim 4, wherein thecontrol zone is comprised as part of one flow-path and the sharedreference zone is comprised as part of the other flow-path.
 8. The assaydevice according to claim 1, wherein the first and second flow-paths arecapable of detecting the presence of an analyte in differentconcentration ranges.
 9. The assay device according to claim 1, whereinthe first and second flow-paths each comprise a porous carriers.
 10. Theassay device according to claim 9, wherein the porous carrier comprisesnitrocellulose.
 11. The assay device according to claim 1, comprising asingle porous sample receiver provided upstream from the first andsecond flow-paths.
 12. The assay device according to claim 1, furthercomprising a sink provided at the distal end of the flow-paths.
 13. Theassay device according to claim 1, wherein the first and secondflowpaths each comprise different amounts of labeled binding reagent.14. The assay device according to claim 1, comprising a first flow-pathfor the detection of an analyte in a lower range and a second flow-pathfor the detection of analyte in a higher range.
 15. The assay deviceaccording to claim 14 wherein the second assay flow-path has a greateramount of labeled binding reagent than the first flow-path.
 16. Theassay device according to claim 1, wherein the first flow-path comprisesa labeled binding reagent for the analyte provided upstream from adetection zone and the second flow-path comprises a labeled bindingreagent for the analyte and a second binding reagent for the analyteprovided upstream from the detection zone.
 17. The assay deviceaccording to claim 16, wherein the first flow-path comprises a sharedreference zone and the second flow-path comprises a shared control zone.18. The assay device according to claim 17, wherein the reference zoneis provided downstream from the detection zone.
 19. The assay deviceaccording to claim 17, wherein the control zone is provided downstreamfrom the detection zone.
 20. The assay device according to claim 1,wherein a photodetector detects light from a plurality of zones.
 21. Theassay device according to claim 20, comprising a single photodetector todetect light from the two detection zones, the reference zone and thecontrol zone.
 22. The assay device according to claim 21, comprisingfour light sources to illuminate the two detection zones, the referencezone and the control zone.
 23. The assay device according to claim 20,wherein the amount of light detected by the photodetector from thedetection zones and the reference zone is measured prior to addition ofsample to the assay device and again after addition of sample to theassay device, and a ratio of the two measurements calculated for eachzone.
 24. The assay device according to claim 20, wherein a normalisedpercentage relative attenuation (% A) is calculated for the detectionand/or control zones wherein: $\; \begin{matrix}{{{HS}_{t}\; \left( {\% \mspace{11mu} A} \right)}{\; \mspace{11mu}} = {\frac{{{Ref}\mspace{11mu} {ratio}_{t}} - {{HS}\mspace{14mu} {test}\mspace{14mu} {ratio}_{t}}}{{Ref}\mspace{11mu} {ratio}_{t}} \times 100\%}} \\{\; {{{LS}_{t}\mspace{11mu} \left( {\% \mspace{11mu} A} \right)}\; = {\frac{{{Ref}\mspace{11mu} {ratio}_{t}} - \; {{LS}\mspace{14mu} {test}\mspace{14mu} {ratio}_{t}}}{{Ref}\mspace{11mu} {ratio}_{t}} \times 100\%}}} \\{{{Ctrl}_{t}\mspace{11mu} \left( {\% \mspace{11mu} A} \right)} = {\frac{{{Ref}\mspace{11mu} {ratio}_{t}} - {{Ctrl}\mspace{11mu} {ratio}_{t}}}{{Ref}\mspace{14mu} {ratio}_{t}} \times 100\%}}\end{matrix}$
 25. The assay device according to claim 1, wherein thelight source comprises one or more LEDs.
 26. The assay device accordingto claim 1, wherein the analyte to be determined is hCG.
 27. the assaydevice according to claim 1, wherein the liquid sample is urine. 28-29.(canceled)